Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity.
The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.
Galizzi
J P
JP
Laboratory for Immunological Research, Dardilly, France.
Castle
B
B
Djossou
O
O
Harada
N
N
Cabrillat
H
H
Yahia
S A
SA
Barrett
R
R
Howard
M
M
Banchereau
J
J
D000818:Animals; D002846:Chromatography, Affinity; D003101:Collodion; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006922:Hylobates; D015847:Interleukin-4 / Q000378:metabolism*; D037102:Lectins; D008970:Molecular Weight; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013601:T-Lymphocytes / Q000032:analysis*; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D037102:Lectins; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D015847:Interleukin-4; D003101:Collodion